This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. The clinically defining feature of type 2 diabetes (T2DM) is hyperglycemia, and as such, glucose metabolism the most widely studied aspect of T2DM. Despite this considerable effort, there remains controversy related to the source of superfluous glucose production in T2DM. Recent work clearly demonstrates that the molecular source of glucose is disturbed in diabetes. In the normal person, after an overnight fast, about 50% of glucose is derived from glycogen and 50% from gluconeogenesis. But in the diabetic person, the gluconeogenic contribution increases to 60%, suggesting a loss of control over this pathway. Notwithstanding this important insight into glucose production in diabetes, there is some disagreement about the anatomical origin of glucose. It is widely accepted that the liver is responsible for nearly all glucose production in the normal post-absorptive state. However, it is also well recognized that other tissues have the capacity to produce glucose in times of need. For instance during the anhepatic phase of liver transplantation in humans or in the total hepatectomy of animals, plasma glucose is maintained by extrahepatic source(s). We've recently shown that in fasted mice lacking hepatic PEPCK, 60% of glucose production comes from tissues other than liver, while the remainder comes from gluconeogenesis from glycerol in liver. Glucose production can be significant in the kidney and small intestine when the whole organism is subjected to compelling conditions such as starvation. Another such compelling condition may occur during acute diabetes where insulin secretion is restricted or the tissues'ability to respond to insulin is impaired. Under these conditions, all glucose producing tissues may respond similarly to that seen during starvation by producing compensatory glucose even though none is needed. While extra-hepatic glucose production has been demonstrated in man, dog and rats after long-term fasting, it may be especially important in mice where the affects of starvation occur more rapidly and are more pronounced. Mice are commonly studied after fasts as long as 24-hours, which may be approximately equivalent to a 72-hour fast in man. This phenomenon must be understood in mice because a growing number of mouse models are used to study human metabolic syndromes and the common assumption that the liver is the only relevant site of gluconeogenesis in mice may not always be true. The kidneys have been recognized as glucose-producing organs for over 60 years, but the significance of their contribution to whole body glucose production remains a subject of debate. Reports of the renal contribution range from 5 - 28% in the post-absorptive state while a 45% contribution was reported after an exhaustive fast of 4-6 weeks. Under certain conditions, renal gluconeogenesis may be doubled in T2DM. The wide range of reported renal contributions to gluconeogenesis reflects how technically difficult it is to make this measurement. Currently, experiment requires catheterization of the renal vein, accurate measurement of blood flow through the kidneys, and determination of A/V differences in glucose concentration. The small intestine represents and additional possibility as a source of glucose production. During insulinopenia the gluconeogenic enzymes PEPCK, pyruvate carboxylase, fructose-1,6-bisphosphatase and glucose-6 phosphatase are up-regulated. It has been estimated that the contribution of the SI to gluconeogenesis may reach 20-25% of total glucose production during diabetes. But like the kidney, estimations of SI gluconeogenesis requires rigorous invasive intervention and the final measurement may carry significant uncertainty. A method which supplies a simple, reliable and clinically relevant indicator of extra-hepatic gluconeogenesis would be extremely helpful to finally understanding the importance of this phenomenon. Hepatic and extra-hepatic glucose productions are difficult to differentiate non-invasively, but the two have distinguishing factors related to the molecular origin of glucose synthesis. For instance, the liver stores large amounts of glycogen while tissues such as the kidneys and small intestine contain only minor amounts of glycogen. Thus their contribution to glucose production must be entirely gluconeogenic rather than glycogenolytic. These two sources are easily distinguished (see preliminary data) by stable isotope tracer methods. The large gluconeogenic contribution in diabetics (60% vs 50% in controls) may be partly due to extra-hepatic glucose production. An important difference between hepatic and extra-hepatic gluconeogenesis is the substrate source. The liver produces glucose largely from lactate and alanine, whereas both the small intestine and kidneys avidly use lactate and glutamine in large preference to alanine. This extra-hepatic preference of glutamine over alanine was recently demonstrated in humans during the anhepatic phase of liver transplantation. We are working to take advantage of this difference in substrate selection to distinguish between hepatic and extra-hepatic contributions to systemic glucose production. These techniques will be tested in the isolated perfused kidney and liver to establish substrate preference and validate in vivo experiments.